Our laboratory recently completed the first comprehensive proteomic analysis of a Gram-negative biofilm matrix. This work demonstrated that a subset of secreted proteins is retained in the biofilm matrix. Three proteins that play a structural role in the biofilm were studied in detail. Interestingly, these proteins were found to have unique distributions in the biofilm matrix that correspond to their functions in biofilm formation. The functions and distributions of several more putative biofilm-associated proteins remain to be characterized. We hypothesize that we can harness these biofilm matrix-associated proteins for use in targeting functional enzymes or antigens to specific regions of the biofilm matrix. Here we propose to develop a toolbox of biofilm matrix association modules and then to use this toolbox in proof of principle experiments in which we associate the secreted enzyme chitinase or the B subunit of cholera toxin with the biofilm matrix in specific distributions. Our goal is to establis a novel strategy for association of proteins with the biofilm matrix of Gram-negative bacteria for use in biomedical, biotechnical, and bioengineering applications of Gram-negative bacteria for use in biomedical, biotechnical, and bioengineering applications. PUBLIC HEALTH RELEVANCE: Here we propose the development of a method for targeting secreted proteins to specific regions of the bacterial biofilm matrix. Such a technology could be useful in biotechnical, bioengineering, and biomedical applications including efficient packaging of secreted antigens in whole cell, killed bacterial vaccines and delivery of functional enzymes to surfaces.